Browsing by Author "Karvonen, Eira"

APECED (Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal-Dystrophy) is a severe, multiorgan autoimmune disease caused by mutations in the AIRE (autoimmune regulator) gene. APECED is a rare disease, however in Finland the frequency is significantly high (1:25 000) and APECED belongs to the ‘Finnish Disease Heritage’. The most common mutation worldwide is the so-called Finn-major mutation R257X that results in a truncation of the AIRE protein, which disrupts the indispensable functions of AIRE. Immune reactions towards body’s own components are typically prevented with various central and peripheral immune tolerance mechanisms. AIRE is essential for the proper development of central and peripheral tolerance and the absence of functional AIRE leads to a loss of immune tolerance and various autoimmune manifestations. Recent studies have suggested that AIRE also has functions in stem cells and actively contributes to the regulation network of pluripotency. Currently, the development of induced pluripotent stem cell (iPSC) technology has opened opportunities for precision medicine and for defining the cure for genetic diseases, such as APECED. The ultimate objective of our research group is to examine whether APECED could be cured via autologous, gene-corrected cell transplants with the use of induced pluripotent stem cells (iPSCs). As a requirement for such later therapeutic use and iPSC differentiation, the APECED patient-derived iPS cells needed to be characterized in detail. To assess, whether AIRE R257X mutation, present in APECED patients’ iPSCs, would cause defects in their stemness properties, the expression of AIRE and classical stem cell markers were examined with qPCR and immunocytochemistry and compared to healthy control iPSCs. The iPSC cells were also treated with spontaneous differentiation -inducing dimethyl sulfoxide (DMSO) to study, whether AIRE R257X mutation would affect the spontaneous differentiation of iPS cells. To further investigate the stemness and early developmental phase properties of APECED patient derived iPSCs, self-aggregated embryoid bodies (EBs) were generated and cultured. Immunocytochemistry was used to examine whether APECED EBs differ in stemness, proliferation or apoptosis from healthy individual’s EBs. The comparative Ct method (ΔΔCt) i.e. fold change revealed that APECED iPSC clones expressed all the classical stem cell markers similarly to healthy control iPSCs. DMSO treatment reduced the expression of stem cell markers in both healthy and APECED-derived iPSCs. The immunostaining results of iPSCs were consistent with the qPCR analysis. The overall growth properties as well as the immunocytochemical assays of stemness, proliferation and apoptosis markers did not show any significant difference between the APECED patient and healthy control derived EBs. Together the results indicate that the R257X mutation of the APECED patients does not affect stem cell properties such as stem cell marker expression and colony or the EB formation of the iPSCs. The results are contrary to previous studies in mice demonstrating the interspecific difference between mouse and human and denoting the importance of human samples completing the studies with animal models. As the APECED patient derived iPSCs did not exhibit any defects in their stemness properties, the later iPS differentiation and therapeutic use could be accomplished without hindrance. However, future work is still needed, as the small sample size in this preliminary test might introduce some biases to the results and hindered a relevant statistical analysis. Nevertheless, this thesis project was the first time APECED patient-derived iPSCs were characterized and has provided new information about the effect of AIRE mutation in APECED patient derived iPSCs.